Fixatives and methods of use

ABSTRACT

Disclosed herein are compositions for fixing tissue for cytologic, histomorphologic, and/or molecular analysis (e.g., DNA, RNA, and/or protein analysis). In some embodiments, the fixatives are aldehyde-free fixatives, for example, formaldehyde- or formalin-free fixatives. Particular disclosed compositions include buffered ethanol. The buffer is a phosphate buffer or phosphate buffered saline (PBS) in some examples. In further embodiments, the fixative includes additional components, such as glycerol and/or acetic acid.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/774,480, filed May 8, 2018, which is the § 371 U.S. National Stage ofInternational Application No. PCT/US2016/061642, filed Nov. 11, 2016,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Application No. 62/255,030, filedNov. 13, 2015, which is incorporated herein by reference in itsentirety.

FIELD

This disclosure relates to preservation of cell or tissue samples,particularly compositions for fixing cell or tissue samples and methodsof their use.

BACKGROUND

Microscopic examination of tissue for diagnostic histopathologicevaluation is most commonly performed on paraffin-embedded tissue. Thisparaffin-embedded tissue is frequently used for molecular diagnostics.The process of tissue embedding (also referred to as impregnation)requires the fixation of the tissue, followed by serial dehydration inalcohols, “clearing” (replacement of the alcohols with an organicsolvent), and replacement of the organic solvent with paraffin (or aresin).

With the advent of molecular pathology, especially RT-PCR, expressionmicroarrays, RNA in situ hybridization, and RNAseq, the interest inobtaining RNA from paraffin-embedded archival tissue has increased.Studies on DNA, including DNA in situ hybridization, PCR, sequencing,mutation detection, genome wide association studies (GWAS), whole exomesequencing, whole genome sequencing and array comparative genomichybridization (aCGH), are frequently applied to paraffin-embeddedtissue. Evaluation of proteins and phospho-proteins may be performed byimmunohistochemistry, or extracted proteins may be evaluated by westernblots, protein array approaches or mass spectroscopy and hybridtechnologies thereof.

The most commonly used fixative is neutral buffered formalin (NBF), asolution typically including 3.7-4% formaldehyde, 5-10% methanol, andphosphate buffer. Concurrent with the health issues of using formalin,this fixative crosslinks proteins, and both nicks and crosslinks RNA andDNA during tissue fixation. These chemical reactions render the primarybiomolecules of interest in tissue damaged, and impairs assays ontissue. Methods to address this damage have been developed for proteins(antigen retrieval) and DNA (Klenow repair); however these onlypartially mitigate the damage. Furthermore, RNA is irreparably damagedduring fixation with NBF and processing.

SUMMARY

There remains an ongoing need to develop fixatives that improve qualityof molecular analysis, particularly analysis of RNA, obtained from fixedsamples. In addition, there is substantial interest in reducing oreliminating the use of formaldehyde, due to its health and environmentalrisks, as well as associated costs and difficulties in disposing ofsolutions containing formaldehyde.

Disclosed herein are compositions for fixing tissue for cytologic,histologic, flow cytometry applications and/or molecular analysis (e.g.,DNA, RNA, and/or protein analysis). In some embodiments, the fixativesare aldehyde-free fixatives, for example, formaldehyde- or formalin-freefixatives. Particular disclosed compositions (also referred to herein as“fixatives” or “fixative solutions”) include buffered ethanol. In someembodiments, the buffer is a phosphate buffer or phosphate bufferedsaline (PBS). In further embodiments, the fixative includes additionalcomponents, such as glycerol and/or acetic acid. In one example, thefixative includes 70% ethanol, PBS, glycerol, and glacial acetic acid. Anon-limiting example of a disclosed fixative is a solution (such as anaqueous solution) including 70% ethanol, 0.5×PBS, 1% glycerol, and 0.5%glacial acetic acid (referred to herein as BE70). The disclosedfixatives are compatible with current standard tissue processingprotocols.

In additional embodiments, the disclosed fixatives include at least onecomponent selected from ethanol, PBS, glycerol and glacial acetic acidand one or more additional components. In some examples, the additionalcomponents include a chaotrope or denaturant (for example, guanidiniumthiocyanate, guanidinium HCl, or guanidinium acetate), trehalose,polyethylene glycol (e.g., PEG200), ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis(β-aminoethyl ether)ethylenediaminetetraacetic acid (EGTA), acrylamide, trichloroaceticacid, acetate salt (e.g., zinc acetate, copper acetate, or magnesiumacetate), acetonitrile, and ethylene glycol. In some examples thedisclosed fixatives are “modular” or “fit for purpose,” e.g., theparticular components are selected based on the planned use of thefixative (such as for applications using nucleic acids (RNA and/or DNA),protein, lipids, electron microscopy (EM), immunofluorescence, orhistochemistry).

Also disclosed herein are compositions for preparation of the disclosedfixatives. In some embodiments, the composition is a “concentrate” suchas an aqueous solution of 1) PBS plus 2) glycerol and/or acetic acidthat is diluted to form a working solution with the desired finalconcentration of each component. In some embodiments, the concentrate isadded to ethanol to arrive at the desired final (working) concentrationof each component. In some examples, the composition is a concentratedsolution for preparation of a disclosed fixative, for example, anaqueous solution containing 1.67×PBS plus 3.33% glycerol and/or 1.67%glacial acetic acid.

Methods of using the fixative solutions are also disclosed herein. Insome embodiments, a biological sample (such as a cell or tissue sample)is immersed or submerged in the fixative under conditions sufficient tofix the sample. In additional embodiments, the fixed sample is processedfor histological, cytological, and/or molecular analysis, for example bydehydrating, clearing, and/or embedding the fixed sample. In someexamples, the fixed (and optionally processed sample) is used formolecular analysis, for example, analysis of one or more biomolecules inthe sample (such as DNA, RNA, and/or protein).

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of digital images of photomicrographs of hematoxylin& eosin (H&E) stained mouse kidney tissue blocks following fixation for24 hours with the indicated fixative and paraffin embedding. Processingconditions for all samples were identical. E, 70% ethanol; EP, 70%ethanol/0.5× phosphate buffered saline; EGP, 70% ethanol/1%glycerol/0.5×PBS; EAP, 70% ethanol/0.5% glacial acetic acid/0.5×PBS;EGAP (BE70), 70% ethanol/1% glycerol/0.5% glacial acetic acid/0.5×PBS;NBF, neutral buffered formalin 10%. These abbreviations are also usedthroughout the remaining figures.

FIG. 2 is a series of digital images of photomicrographs ofrepresentative immunohistochemical images of AQP1 and CD31 staining ofsections from mouse kidney tissue blocks after fixation with theindicated fixative and paraffin embedding.

FIGS. 3A and 3B are a pair of graphs showing protein quantity andquality obtained after mouse kidney fixation with the indicated fixativeand paraffin embedding. FIG. 3A shows the amount of protein extractedfrom each condition, measured using BCA Protein Assay Kit. The proteinextraction yield was expressed the mean of three replicated samples(mean±SD). FIG. 3B shows protein integrity of different fixativesolutions assessed by Western blotting (in triplicate, quantified bydensitometry). Inset, digital image of representative Western blot.Relative GAPDH signal of each entity was normalized to NBF-fixedsamples.

FIGS. 4A-4C are a series of panels showing RNA quantity and qualityobtained after mouse kidney fixation with the indicated fixative andparaffin embedding. FIG. 4A is a graph showing amount of RNA extractedfrom each specimen measured by the Nanodrop spectrophotometer. The RNAextraction yield was expressed the mean of three replicated samples(mean±SD). FIG. 4B is a representative electropherogram showing anoverlay of all seven different conditions. Inset shows an enlargement ofthe area enclosed in dotted lines. FF, fresh frozen mouse kidney(positive control). FIG. 4C is a graph showing integrity value of RNApresented as RNA quality metric (QM).

FIGS. 5A-5D are a series of panels showing RNA integrity profile of RNAsamples derived from mouse kidney tissues, fixed with the indicatedfixative and paraffin embedded. The gene expression profile of tumornecrosis factor signaling genes was analyzed by multiplex reversetranscription-polymerase reaction (RT-PCR) using the MPCR kit (MaximBiotech). An aliquot (1 μl) of the PCR reaction was run on the Agilent2100 Bioanalyzer using DNA 1000 chip. Representative data are shown as agel-like image (FIG. 5A) and an electropherogram (FIG. 5B). Fixativeconditions of each sample in FIG. 5A are indicated above each lane asfollows: 1, E; 2, EP; 3, EGP; 4, EAP; 5, EGAP; 6, NBF; F, fresh frozenmouse kidney; P, positive control; N, negative control (water); M,molecular weight marker. To compare the quality of PCR amplicons, FIG.5B shows an electropherogram overlay of EGAP and NBF fixative condition.Each symbol represents the difference between EGAP and NBF. The symbolscorrespond to the bands marked by the same symbol in FIG. 5A. FIG. 5C isa graph showing 18S rRNA expression levels as a box plot. The values arethe average quantitative real-time RT-PCR cycle threshold numbers(Ct-values). The bars indicate standard deviation (n=3). FIG. 5D is agraph showing HPRT expression levels as a box plot. The values are theaverage quantitative real-time RT-PCR cycle threshold numbers(Ct-values). The bars indicate standard deviation. *, p<0.05; **,p<0.01; ***, p<0.001 (FIGS. 5C and 5D).

FIGS. 6A-6C are a series of panels showing DNA quantity and qualityobtained from mouse kidney fixed with the indicated fixative solutionsand embedded in paraffin. FIG. 6A is a graph showing amount of DNAextracted from each specimen is measured by the Nanodropspectrophotometer. The DNA extraction yield was expressed the mean ofthree replicated samples (mean±SD). FIG. 6B is a graph showing DNAquality assessed by BioScore™ Screening and amplification kit. FIG. 6Cis a graph showing average cycle threshold (Ct) values of a housekeepinggene (HPRT) tested in kidney under different fixative conditions. Geneexpression levels are shown as box plot. The values are the average PCRCt-values. The bars indicate standard deviation. *, p<0.05; **, p<0.01;***, p<0.001.

FIGS. 7A and 7B are a series of panels showing immunohistochemistry ofmouse kidney and mouse spleen, respectively, fixed with BE70 or NBF forthe indicated amounts of time. FIG. 7A shows staining of Aquaporinantibody in kidney and FIG. 7B shows staining of Ki67 in spleen.

FIGS. 8A-8C are a series of panels showing protein quantity and westernblotting in kidney tissue fixed with BE70 or NBF for the indicatedamounts of time. FIG. 8A shows total protein recovery. The data isnormalized to the quantity of protein recovered from tissue fixed withBE70 for one day. FIGS. 8B and 8C show western blotting (top) andquantitation of the western blot data (bottom) for AKT (FIG. 8B) andGAPDH (FIG. 8C) for different fixation times with ethanol, BE70, or NBF.

FIG. 9 is a series of panels showing H&E staining of mouse liver tissuefixed with BE70 or NBF for the indicated amounts of time.

FIG. 10 is a digital image of a western blot for AKT and GAPDH in kidneytissue fixed with the indicated fixative for 24 hours.

FIG. 11 is a graph showing paraffin embedded RNA metric (PERM) forkidney tissue fixed with the indicated fixative for 24 hours.

FIGS. 12A-12C are a series of graphs showing cycle threshold of TLR-4 inmouse liver (FIG. 12A), kidney (FIG. 12B), or liver+kidney (FIG. 12C)tissue fixed with the indicated solutions.

FIGS. 13A-13C are a series of graphs showing cycle threshold of ACTB inmouse liver (FIG. 12A), kidney (FIG. 12B), or liver+kidney (FIG. 12C)tissue fixed with the indicated solutions.

FIGS. 14A and 14B are panels showing Western blotting of AKT and GAPDHin mouse liver or kidney samples fixed with the indicated fixatives(FIG. 14A) or total protein recovery in mouse liver or kidney samplesfixed with the indicated fixative (FIG. 14B).

DETAILED DESCRIPTION

Disclosed herein are buffered ethanol-containing fixatives that provideadvantages over aldehyde-containing fixatives (such as NBF),particularly with respect to quality of nucleic acids (RNA and DNA) andproteins recovered from fixed and embedded tissues. With reference tocytologic and histomorphologic features, the disclosed buffered ethanolfixatives offer less shrinkage than unbuffered alcoholic fixatives. Withrespect to cytology and histomorphology, it is well known that formalingenerates “reproducible artifacts” that are often used by pathologistsin their diagnosis, and these differences may not be present withethanol fixatives. The fixatives disclosed herein, particularly BE70,also generate a “reproducible artifact;” however, this artifact may notbe identical to that imparted by NBF. In some examples, inclusion ofglacial acetic acid in a buffered ethanol fixative may offset some ofthese differences, as it appears to add “crispness” to the cytologicfeatures.

Immunohistochemistry on tissue fixed with the disclosed buffered ethanolfixatives can be optimized by routine methods and generates stainingpatterns of functionally identical features compared to NBF. Generally,non-crosslinking fixatives require less antigen retrieval than NBF fixedtissue, and tissue fixed with the buffered ethanol formulationsdisclosed herein follow this pattern. Some antigens may not bedetectable in tissues fixed with buffered ethanol, e.g., with antibodiesthat have been selected for detection by immunohistochemistry on NBFfixed tissue. In these instances, dipping the deparaffinized slide inNBF for 10 seconds prior to antigen retrieval is commonly used to inducesufficient formalin-mediated chemical changes as to make the antigendetectable (see, e.g., Panzacchi et al., Eur. J. Oncol. 18:75-83, 2013).In some examples, the dipping of deparaffinized slides in NBF prior toantigen retrieval may not be necessary for samples fixed using thefixatives disclosed herein. This may be antigen-dependent and can bedetermined utilizing routine testing.

In addition to providing improved quality of cytologic,histomorphologic, and molecular analyses compared to NBF or otherformaldehyde containing fixatives, the disclosed buffered ethanolfixatives provide improved ease of use and stability. Furthermore,because they do not contain formaldehyde, the disclosed fixativespresent reduced hazards to personnel and the environment and are lessexpensive to dispose of than aldehyde-containing fixatives. In addition,the disclosed fixatives are not damaging to tissue processors, are notflammable, and are compatible with standard tissue processing protocols.Thus, the disclosed fixatives can be used as a direct replacement forNBF in tissue processing.

I. ABBREVIATIONS

-   -   E 70% ethanol    -   EAP 70% ethanol/0.5% glacial acetic acid/0.5×PBS    -   EGAP 70% ethanol/1% glycerol/0.5% glacial acetic acid/0.5×PBS,        also referred to as BE70    -   EGP 70% ethanol/1% glycerol/0.5×PBS    -   EP 70% ethanol/0.5×PBS    -   H&E hematoxylin and eosin    -   NBF neutral buffered formalin    -   PBS phosphate buffered saline    -   QM RNA quality metric    -   RIN RNA integrity number

II. TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and George P. Rédei, EncyclopedicDictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003(ISBN: 0-471-26821-6).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Although methodsand materials similar or equivalent to those described herein can beused in the practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, pH, temperatures, times, andso forth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that can depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Aldehyde-free: Lacking or substantially lacking presence of one or morealdehyde-containing compounds (such as formaldehyde, paraformaldehyde,or glutaraldehyde), for example, containing 0.5% or less of analdehyde-containing compound. In some examples, aldehyde-free indicatesthat a composition lacks added or exogenous aldehyde-containingcompounds (but for example, may include some amount of endogenousaldehyde-containing compounds).

Embedding medium: A substance in which tissue, such as fixed anddehydrated tissue is placed or enclosed. Exemplary embedding mediainclude paraffin and resins, such as paraffin, paraffin-containingcompounds, araldite, celloidin, Durcupan™, epoxy, glycol methacrylate,hydroxypropyl methacrylate, JB-4™, Spurr, LR White™, polyester, andpolyethylene glycols.

Fixative: An agent or combination of agents that act as a preservative,for example, that renders an end to endogenous biologic processes (suchas respiration and glycolysis) in cells or tissue. In addition, afixative inhibits degradation (e.g. putrification), inhibits growth ofbacteria, and renders the cells or tissue non-infectious. Fixatives canbe used to preserve fresh tissue for subsequent examination (such ashistology or molecular analysis).

Neutral buffered formalin (NBF): Also referred to as 10% neutralbuffered formalin. A solution including approximately 3.7-4.0%formaldehyde in a buffer, typically a phosphate solution, of mono-basicand dibasic phosphate. NBF typically also includes 5-10% methanol (see,e.g., Fox et al., J. Histochem. Cytochem. 33:845-853, 1985).

Phosphate buffered saline (PBS): A solution containing sodium phosphateand sodium chloride, and typically also potassium phosphate andpotassium chloride and having a pH of about 7.4. In particularembodiments, 1×PBS is a solution containing 137 mM NaCl, 2.7 mM KCl, 4.3mM Na₂HPO₄, and 1.47 mM KH₂PO₄, with pH 7.4. In another example, 1×PBSis a solution containing 1.7 mM KH₂PO₄, 5 mM Na₂HPO₄, 150 mM NaCl, pH7.4. However, one of ordinary skill in the art will recognize thatformulations for PBS vary and may not include KCl and/or KH₂PO₄, or maycontain varying concentrations of the components. For example, in somecases, 1×PBS may contain about 150 mM NaCl. In its simplest formulation,1×PBS contains about 10 mM phosphate and about 135-155 mM NaCl at pH7.2-7.5. Optionally, PBS may also contain CaCl₂ and MgCl₂ (e.g., 1.33 gCaCl₂.2H₂O and 1.0 g MgCl₂.6H₂O) in 1 liter of 1×PBS.

In some examples, PBS is prepared as a concentrated stock (such as 2×,5×, 10×, or 20×) and is diluted with water to the desired concentration(such as 0.1×, 0.5×, or 1×) prior to use. PBS (for example in aconcentrated stock solution, such as 10×PBS) is also commerciallyavailable, for example from Thermo Fisher Scientific (Waltham, Mass.) orSigma-Aldrich (St. Louis, Mo.).

Sample: A biological specimen containing DNA, RNA (including mRNA),protein, or combinations thereof, obtained from a subject. Examplesinclude, but are not limited to, peripheral blood, fine needle aspirate,cells (such as a cytology smear), and tissue. In some examples, “tissuesample” includes whole organs or a portion thereof, organsub-structures, surgical tissue biopsies, punch biopsies, fine-needleaspirate biopsies, bone, biological fluids, archival tissues, or cells(including cells obtained from a subject or cells grown or cultured invitro). The subject from which a sample is obtained includes anysingle-celled organism (e.g., bacteria or fungus), plant, or vertebrateorganism (including but not limited to human, non-human primate,veterinary or laboratory animal, rodent, horse, sheep, cow, pig, bird,reptile, or amphibian).

III. FIXATIVE SOLUTIONS

Disclosed herein are compositions for fixing tissue, for example, forcytologic, histologic, flow cytometry and/or molecular analysis (e.g.,DNA, RNA, and/or protein analysis). In some embodiments, the disclosedcompositions (referred to herein as “fixatives” or “fixative solutions”)include buffered ethanol. In some embodiments, the fixatives arealdehyde-free or substantially aldehyde-free fixatives, for example,fixatives that contain 0.5% or less aldehydes and/or that do not includeadded formaldehyde, formalin, paraformaldehyde, glutaraldehyde, or otheraldehyde-containing compounds. In additional embodiments, the fixativesare non-crosslinking fixatives, for example, fixatives that do notcrosslink protein and/or do not result in substantial nicking orcrosslinking of RNA or DNA. In the description of the fixative solutionsprovided herein, all amounts are by volume (v/v).

The disclosed fixatives include ethanol and a buffer. In someembodiments, the fixatives also include additional components, such asglycerol and/or acetic acid. In particular examples, the fixativesdisclosed herein produce less tissue shrinkage and hardening than isobserved in tissues fixed with 70% ethanol and produce tissuehistomorphology that is functionally comparable to that obtained withthe benchmark NBF. Furthermore, tissue fixed with the disclosed bufferedethanol fixatives result in improved RNA recovery, both in terms ofquantity of RNA, as well as higher RNA quality (for example, recovery oflonger RNA fragments) than is observed with tissue fixed with 70%ethanol alone.

In addition, the disclosed fixatives provide improved qualities whencompared to NBF. In some examples, tissue fixed with the fixativesdisclosed herein including ethanol and a buffer provide improved RNAquality (for example, as reflected by RNA quantity or fragment length,or as measured by RNA quality metric (QM) or RNA integrity number (RIN))than tissues fixed under the same conditions with NBF. In additionalexamples, tissues fixed with the fixatives disclosed herein includingethanol and a buffer result in more sensitive detection of RNA or DNA(for example, based on cycle threshold in real-time PCR) than tissuesfixed under the same conditions with NBF. In further examples, thedisclosed fixatives provide substantially identical or improved tissuestaining results (for example, improved hematoxylin staining) comparedto tissue fixed with NBF. In a specific example, and without being boundby theory, it is believed that the buffering and pH range of thedisclosed fixatives normalizes the intensity of hematoxylin stainingacross different tissues. In addition, it is believed that improvedhistology (preservation) of tissue contributes to the improved stainingcharacteristics imparted by the disclosed fixatives.

In particular embodiments, the fixative includes 70-80% ethanol and abuffer (for example, a phosphate buffer or PBS). In some examples, thefixative includes 70-75% ethanol, 73-78% ethanol, or 76-80% ethanol. Inparticular examples, the fixative includes 70% ethanol. In someexamples, the buffer is PBS (e.g., 1×PBS is 1.7 mM KH₂PO₄, 5 mM Na₂HPO₄,150 mM NaCl, pH 7.4 or 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.47mM KH₂PO₄, pH 7.4, though other variations of PBS can also be used). Theamount of PBS included in the fixative solution ranges from 0.1×-1×, forexample 0.2×-0.8×, 0.1-0.5×, or 0.4-1×. In particular examples, theamount of PBS in the fixative solution is 0.1×, 0.2×, 0.4×, 0.5×, 0.6×,0.8×, or 1×. One specific non-limiting example of a fixative disclosedherein contains 70% ethanol and 0.5×PBS.

Without being bound by theory, it is believed that including saline inthe fixative (e.g., in the form of PBS) contributes to cytomorphologyquality, such as reducing cell shrinkage, most likely by exertingosmotic effects. However, saline is not required in the buffered ethanolfixatives disclosed herein. Buffers that do not include saline (such asa phosphate buffer without saline) can also be used. In other examples,a buffer that does not include phosphate is used, such as Tris, CaCl₂,2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), or3-(N-morpholino)propanesulfonic acid (MOPS).

The pH of the fixatives disclosed herein have a final pH of 5 to 8, forexample, pH 5-6, 5.5-6.5, 6-7, 6.5-7.5, 6.5-7, 7-7.5, or 7-8. Forexample, the pH can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8. In some non-limiting examples, thefixatives have a pH of 6.5. In other non-limiting examples, thefixatives have a pH of 7. In further non-limiting examples, the pH ofthe fixatives is 6.1. In some examples, the pH of the fixative isadjusted (for example, with NaOH) following mixing of the components toachieve the desired final pH.

In further embodiments, the buffered ethanol fixative includesadditional components. In some examples, the additional componentsinclude glycerol and/or acetic acid.

In some embodiments, the fixative includes ethanol, a buffer andglycerol. In particular examples, the fixative includes ethanol and aphosphate buffer (such as PBS) in any combination of amounts asdescribed above, plus 0.1-10% glycerol, such as 0.2-5%, 0.5-3%, 0.1-2%,1-5%, 2-6%, or 3-10%. In some examples, the amount of glycerol in thefixative is 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2.5%, 5%, 7.5%, or 10%. In someexamples, the fixative includes 70% ethanol, 0.1×-1×PBS, and 0.1-10%glycerol. In one specific non-limiting example, the fixative includes70% ethanol, 0.5×PBS, and 1% glycerol. Without being bound by theory,presence of glycerol in the disclosed fixatives may improve cellmembrane penetration of the fixative during tissue fixation. However,inclusion of glycerol is not required in the fixatives disclosed herein.

In other embodiments, the fixative includes ethanol, a buffer, and anacid (such as glacial acetic acid or picric acid). In particularexamples, the fixative includes ethanol and a phosphate buffer (such asPBS) in any combination of amounts as described above, plus 0.1-10%acetic acid, such as 0.1-5%, 0.5-3%, 1-10%, 0.5-2.5%, 2-6%, 5-7.5%, or4-10%. In some examples, the amount of acetic acid in the fixative is0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, or 5%. In someexamples, the fixative includes 0.5% glacial acetic acid. In someexamples, the fixative includes 70% ethanol, 0.1×-1×PBS, and 0.1-5%glacial acetic acid. In one specific and non-limiting example, thefixative includes 70% ethanol, 0.5×PBS, and 0.5% glacial acetic acid.Without being bound by theory, presence of acetic acid in the disclosedfixatives may provide improved (e.g. clearer or sharper) histomorphologyand/or enhanced cell penetration of the fixative. The fixativesincluding acetic acid disclosed herein may also provide improved qualityof DNA recovered from fixed tissue. However, inclusion of acetic acid(or other acid) is not required in the fixatives disclosed herein.

In still further embodiments, the fixative includes ethanol, a buffer,glacial acetic acid, and glycerol. In particular examples, the fixativeincludes ethanol, buffer (such as phosphate buffer, for example PBS),glacial acetic acid, and glycerol in any combination of amounts asdescribed above or shown in Tables 1-3. In some examples, the fixativeincludes 70% ethanol, 0.1×-1×PBS, 0.1-10% glycerol, and 0.1-5% glacialacetic acid. In one non-limiting example, the fixative includes orconsists of 70% ethanol, 0.5×PBS, 1% glycerol, and 0.5% glacial aceticacid.

Also disclosed are additional embodiments of the fixatives that includealterations to one or more of the components (e.g., an increased ordecreased amount), inclusion of one or more additional components, orboth. In some embodiments, the disclosed fixatives may be modular, e.g.,one or more components may be added or removed depending on the intendedpurpose or use of the fixed cells or tissues (such as RNA or DNArecovery and analysis, protein recovery and analysis, flow cytometry,electron microscopy, histomorphology, immunofluorescence, and so on).Table 1 sets out various components that can be included in a fixativecomposition. In certain embodiments, at least one of the additionalcomponents could be added to a base fixative that includes ethanol andPBS. In certain embodiments, the base fixative is particularly 70%ethanol and 0.5×PBS. Paraformaldehyde and/or glutaraldehyde are onlyused as fixative additives for purposes of EM and infectious diseasepathology.

Guanidinium salt(s) may replace “unfreezable/non-freezable water” withprotection of RNA, DNA and protein via hydrogen bonds. Guanidinium, byreplacing “unfreezable/non-freezable water”, may improve biologicalspecimen stability, by reducing the opportunity for hydrolysis andoxidation of nucleic acids and proteins. See Boi, Scalia, Gendusa,Ronchi, and Cattoretti, Disaccharides Protect Antigens fromDrying-Induced Damage in Routinely Processed Tissue Sections, JHistochem Cytochem January 2016 64: 18-31, for an explanation of“non-freezable water”. Illustrative guanidinium salts includeguanidinium HCl, guanidinium thiocyanate, guanidinium acetate,guanidinium sulfate, guanidinium nitrate, guanidinium nitrate,guanidinium carbonate, guanidinium formate and guanidinium phosphate.

TABLE 1 Exemplary fixative components and their use in specificapplications of the fixed cells or tissue Amount in illustrativeSpecific Additive embodiment Impact Application Water — — Ethanol 70% —— EDTA or EGTA Saturation Remove 1. Decalcification Divalent Ions 2. DNAIsolation Glycerol Up to 10% Improved Base Fixative in Biomolecularcertain Recovery embodiments Acetic Acid Up to 7% Cell Increases Rate ofPenetration Cell Penetration (Flow Cytometry) NaOH raise pH — PBSconcentrate 0.5X osmotic off-sets shrinkage balance; of high osmolyteconcentrations of EtOH, and improves access to proteins aftercoagulation Guanidinium Saturation chaotrope improved nucleic Saltsacids and proteins Trehalose 0.1%- chaotrope Improved nucleic saturationacids Acrylamide Up To 0.4% matrix EM Polyethylene 0.1%- matrix EMGlycol (200 MW) saturation Trichloroacetic Less than Cellular EM Acid(TCA) 25% penetration Acetate 0.1%- Cell Flow Cytometry; saturationpenetration EM Acetonitrile 0.1%- cellular EM saturation penetrationEthylene Glycol 0.1%- cellular protein saturation penetrationparaformaldehyde Saturation crosslinking EM & Infectious DiseasePathology glutaraldehyde Saturation crosslinking EM & Infectious DiseasePathology

The disclosed fixatives are stable at room temperature for at least 3months and do not precipitate at 4° C. for at least one month, makingthem suitable for laboratory storage. Furthermore, if precipitatedevelops upon storage at 4° C., the precipitate goes back into solutionupon mixing. In some examples, the fixative is stable at 4° C. for atleast 3 months, at least 6 months, at least 12 months, or more.

In additional embodiments, disclosed herein are compositions thatinclude each of the fixative components except for ethanol, for example,prepared as a concentrate. The final (“working”) fixative solution isprepared by adding the concentrate to ethanol to provide the desiredfinal concentration of ethanol and the other components. The concentrateis stable at room temperature and 4° C., making it suitable forlaboratory storage and providing convenient usage by preparing workingsolutions as needed. In some cases, the concentrate develops aprecipitate upon storage at 4° C., but the precipitate easily goes backinto solution and does not affect performance. In some examples, theconcentrate is stable at 4° C. for at least 3 months, at least 6 months,at least 12 months, or more.

Thus, in some embodiments, the disclosure provides compositions (e.g.,concentrates) including phosphate buffer (for example, PBS) plusglycerol and/or glacial acetic acid in a more concentrated form than inthe fixative solutions disclosed herein. For example, the concentrateincludes each of the components combined in an aqueous solution suchthat it can be added to ethanol (such as 100% ethanol or 95% ethanol) toproduce the desired final concentration of the fixative components. Forexample, the components can be formulated as a solution such that about25-30 ml of the concentrate, when added to 95%-100% ethanol to a finalvolume of 95-100 ml, produces a solution with the desired finalconcentration of the components, including 70% ethanol. Thus, in onenon-limiting example, the concentrate includes or consists of 1.67×PBS,3.33% glycerol, and 1.67% glacial acetic acid in an aqueous solution. Inthis example, a working solution is prepared by mixing 30 ml of thestock solution with 100% ethanol to arrive at a volume of 100 ml (givinga final concentration of 70% ethanol in the solution). In some examples,the concentrate is prepared by combining PBS (such as 10×PBS), glycerol(such as 10% (v/v) glycerol), and glacial acetic acid in the listedorder, followed by addition of water to produce a concentrate with thedesired concentration of each component. The pH of the concentrate canbe adjusted after mixing the PBS, glycerol, and glacial acetic acid (forexample, prior to adding water), to achieve a desired pH. In oneexample, the pH of the concentrate is 4.3. One of ordinary skill in theart can produce concentrates for preparation of any of the fixativesdisclosed herein.

IV. METHODS OF TISSUE FIXATION

Methods of fixing tissue with the fixative solutions are providedherein. In some embodiments, the methods include immersing or submerginga biological sample in a fixative solution disclosed herein. Exemplarysamples that can be used in the methods disclosed herein include, butare not limited to, whole organs or a portion thereof, organsub-structures, surgical tissue biopsies, punch biopsies, fine-needleaspirate biopsies, bone, archival tissues, or cells. In other examples,samples include specimens after dissection by a pathologist orlaboratory technician, and in some instances immersed in fixative,dissected (e.g., sectioned) and then re-immersed in fixative. In someexamples, these biological samples are referred to as “tissues” or“tissue samples.” In cases where a sample is large, it can be cut intosmaller pieces (such as pieces 2 mm thick or less), for ease of handlingand improved fixative penetration. In other examples, a large specimenis immersed in the fixative whole, and remains immersed for a suitableperiod of time for the specimen to be fixed (e.g., days or weeks).

In some embodiments, the methods include immersing (for examplecompletely covering) a sample with a fixative solution disclosed hereinunder conditions sufficient to fix the sample. In some examples, thetissue or sample is immersed in a 10- to 20-fold excess volume offixative solution; however, this can be varied, for example from “equalvolume” fixation (1:1 by fluid displacement) to greater than 100-foldexcess volume. The tissue remains immersed in the fixative solution fora sufficient time to fix the tissue, for example at least 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48hours, or more (such as 2-12 hours, 4-18 hours, 6-72 hours, 8-48 hours,12-24 hours, or 16-36 hours). In particular examples, the sample isimmersed in the fixative for about 24 hours. However, one of ordinaryskill in the art can determine suitable fixation times for particulartypes and sizes of specimens using routine testing methods.

The fixation is carried out a 4° C.-45° C., such as at 4° C., roomtemperature (e.g., about 20-25° C.), 30° C., 37° C., or 45° C. (forexamples, 4-12° C., 18-25° C., 30-45° C., 32-42° C.). In other examples,the fixation is accelerated by microwave or ultrasound. In specificexamples, the fixation is carried out at room temperature. In oneparticular example, the sample is fixed by immersing the sample in afixative disclosed herein for 24 hours at room temperature. In furtherembodiments, fixation can be as short as 4 hours and as long as onemonth, depending of the assay, without degradation of the biomoleculesand morphology. One of ordinary skill in the art can determine fixationconditions (such as time and/or temperature of fixation) for aparticular sample or sample type utilizing routine methods.

In some embodiments, following fixation, the sample is processed andembedded according to standard protocols, including dehydration (forexample, through a series of graded ethanols), clearing, and embeddingin paraffin or another embedding medium (such as araldite, celloidin,Durcupan™ epoxy, glycol methacrylate, hydroxypropyl methacrylate, JB-4™,Spurr, LR White™, polyester, or PEGs). These steps are performedaccording to standard histological techniques, and can be carried outusing an automated tissue processor (such as a Tissue-Tek processor,Sakura Finetek, Torrance, Calif.), manual bench-top processing, ormicrowave processing. One of ordinary skill in the art can select tissueprocessing and embedding conditions for particular tissues and desireddownstream use with routine experimentation. In some examples, theembedded tissue is sectioned, for use or storage, while in otherexamples, embedded tissue blocks are placed in storage for later use. Inother examples, the fixatives disclosed herein are also suitable for useas a cytology fixative (e.g., without impregnation). For example,following fixation, the sample is moved to alcohol, then smeared or runthrough a liquid cytology system. Alternatively, cells are smeared ontoa slide, which is then dipped in fixative, and subsequently stained.

In additional embodiments, the fixed, embedded tissue or tissue sectionis utilized for cytology, histomorphologic, flow cytometry or molecularanalysis. Thus, in some examples, tissue samples are stained, forexample with hematoxylin and eosin (H&E) or other histology stains. Inother examples, the tissue samples or sections are utilized for in situhybridization or immunohistochemistry. In still further examples,nucleic acids (such as RNA or DNA), or protein are extracted from thetissue samples or sections and utilized for analyses such as PCR,RT-PCR, real-time PCR or RT-PCR, quantitative real-time PCR or RT-PCR,microarray analysis, sequencing, Southern blotting, Northern blotting,or Western blotting. Exemplary histomorphologic and molecular analysismethods are described below. One of ordinary skill in the art can selectthese or other analytical methods suitable to the particular tissue andpurpose (such as diagnosis of a particular disease or condition).

EXAMPLES

The following examples are illustrative of disclosed embodiments. Inlight of this disclosure, those of skill in the art will recognize thatvariations of these examples and other examples of the disclosedtechnology would be possible without undue experimentation.

Example 1 Materials and Methods

Fixatives: Six different fixatives were tested: 70% ethanol (E), 70%ethanol+0.5×PBS (EP), 70% ethanol+1% glycerol+0.5×PBS (EGP), 70%ethanol+0.5% glacial acetic acid+0.5×PBS (EAP), 70% ethanol+1%glycerol+0.5% glacial acetic acid+0.5×PBS (EGAP or BE70), and neutralbuffered formaldehyde 10% (NBF). The final formulation of fixative wasadjusted by volume. Glycerol and glacial acetic acid were purchased fromSigma-Aldrich (St. Louis, Mo.). Ethanol and NBF were purchased from VWR(Radnor, Pa.). 1×PBS was 1.7 mM KH₂PO₄, 5 mM Na₂HPO₄, 150 mM NaCl, pH7.4. All fixatives were stored and used at room temperature.

Tissue Samples and Fixation: Mouse specimens were acquired from theNational Institutes of Health (NIH), Small Animals Section, VeterinaryResources Branch. The animals were housed and euthanized in accordancewith NIH guidelines for care and use of laboratory animals. The ischemiatime periods were quite similar (2-3 min) for the different samples. Inorder to examine the impact of the fixatives in comparison to 10% NBF inantigen degradation and molecular quality, mouse kidney samples werefixed for 24 hours at room temperature in 10 ml of the differentfixatives. Tissues were then processed using an enclosed automatedprocessor (Tissue-Tek VIP IV, Sakura Finetek Inc., Torrance, USA) atroughly 30-45 minutes per station. Briefly, tissues were dehydrated in aseries of ethanol, then cleared with xylene prior to infiltration withmolten paraffin. Tissues were then embedded in paraffin and sectionedfor histological and molecular evaluation. Processing has also beencarried out in other instruments, including anethanol/isopropyl/paraffin sequence, with and without microwave. Allgave the same results with reference to fixative performance.

Immunohistochemical and Histochemical Evaluation: Immunohistochemicalstaining was performed on 5 micron thick sections. The tissue sectionswere deparaffinized through xylene and dehydrated with graded ethanol.Endogenous peroxidase activity was quenched with 3% H₂O₂ in water for 10minutes. Additional blocking to minimize non-specific staining was donewith Protein Block solution (Dako, Carpinteria, Calif.) for 15 minutes.After washing with TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween®20), the slides were incubated with rabbit anti-aquaporin 1 (AQP1)polyclonal antibodies (Cat. #20810; dilution 1:250; Santa CruzBiotechnology, Santa Cruz, Calif.) or rabbit anti-CD31 polyclonalantibodies (Cat. #ab28364; dilution 1:50; Abcam, Cambridge, Mass.) for30 minutes at room temperature. Antigen-antibody reaction was detectedusing an EnVision™+peroxidase kit and visualized with3,3-diaminobenzadine (Dako). Slides were then lightly counterstainedwith hematoxylin, dehydrated in ethanols, cleared in xylene, andcoverslipped. Stained slides were observed under a light microscope(Axioplot, Carl Zeiss, Jena, Germany). Hematoxylin and eosin (H&E) stainwas done concurrently with each tissue sample to examinehistomorphological features. The stained sections were digitizedutilizing a NanoZoomer® 2.0 HT slide scanner (Hamamatsu Photonics K.K.,Japan) at 40× objective magnification (0.23 μm/pixels resolution).

Protein Extraction and Western Blotting: Protein extraction from two 10μm formalin fixed paraffin-embedded (FFPE) tissue sections was performedas previously described (Chung et al., Proteomics Clin. Appl.2:1539-1547, 2008). Briefly, sections were trimmed of excess wax andhomogenized using a Disposable Pellet Mixer in 200 μl protein extractionsolution (1× high pH Antigen retrieval buffer (pH 9.9) (Dako), 1% NaN₃,1% SDS, 10% glycerol and protease inhibitor (1 tablet/25 ml, Roche)),followed by incubation for 15 minutes at 115° C. within a pressurecooker (Dako). After incubation, the tissue lysates were centrifuged at13,000 rpm for 30 minutes at 4° C. The supernatants were collected andstored at −20° C. Total protein concentrations were measured with theBCA Protein Assay kit (Pierce Biotechnology, Rockford, Ill.).

Ten micrograms of total protein extracted from different fixativesolutions were resolved by 4-12% NuPAGE® Bis-Tris polyacrylamide gel(Life Technologies, Grand Island, N.Y.), and transferred tonitrocellulose membrane using iBlot™ Dry Blotting System (Invitrogen,Carlsbad, Calif.). The membranes were blocked with 5% nonfat dry milk inTBST for 1 hour, washed, and subsequently incubated overnight at 4° C.in TBST with mouse anti-GAPDH monoclonal antibodies (clone 6C5; dilution1:3000; Calbiochem, Gibbstown, N.J.). Specific molecules were detectedwith horseradish peroxidase-labeled anti-mouse secondary antibodies(Chemicon International, Temecula, Calif.) and enhanced withSuperSignal™ Chemiluminescence kit (Pierce Biotechnology). Signals weredetected on BioMax® MR X-ray film (Kodak, Rochester, N.Y.). Quantitativeanalysis of the western blotting was performed using ImageQuant®software (Ver. 5.2, Molecular Dynamics, Sunnyvale, Calif.).

RNA Extraction and cDNA Synthesis: RNA extraction from two 10 μm tissuesections was performed as described previously (Chung et al., Diagn.Mol. Pathol. 15:229-236, 2006). Briefly, sections were trimmed of excesswax and deparaffinized by three incubations in PROTOCOL buffer (FisherScientific; Kalamazoo, Mich.) for 15 minutes at 95° C. with shaking,followed by three centrifugations at room temperature for 2 min at10,000×g. Subsequently, specimens were briefly rinsed once in 100%ethanol. The sections were resuspended and ground in a solution of 4 Mguanidine isothiocyanate, 20 mM sodium acetate, and 25 mMβ-mercaptoethanol (pH 5.5), followed by incubation for 72 hours at 65°C. with mild shaking. After incubation, RNA was isolated byphenol/chloroform extraction. In order to remove possible contaminatinggenomic DNA, the extracted RNA was treated with 2 μl TURBO™ DNasebuffer, 4 units TURBO™ DNase (Invitrogen, Carlsbad, Calif.) and 40 unitsof RNase inhibitor (Promega; Madison, Wis.) in a 100 μl reaction volume.The mixture was incubated at 37° C. for 30 minutes, followed bypurification with phenol/chloroform extraction. For frozen tissue, totalRNA was obtained using TRIzol® reagent (Invitrogen) and further purifiedwith RNeasy® minikits (Qiagen, Valencia, Calif.), according to themanufacturer's instructions (Chung et al., J. Histochem. Cytochem.56:1033-1042, 2008).

Approximately 5 μg of total RNA for each sample was transcribed intocomplementary DNA (cDNA). Extracted RNA, random hexamers (Promega), andSuperScript® II RT kit (Invitrogen) were used to synthesize the cDNA(Chung et al., Diagn. Mol. Pathol. 15:229-236, 2006; Chung et al., J.Histochem. Cytochem. 56:1033-1042, 2008). All samples were reversetranscribed under the same conditions. The synthesized cDNA was storedat −20° C. and used as a template in multiplex reversetranscription-polymerase chain reaction (RT-PCR) reactions.

RNA Quantity and Quality: The amount of RNA was determined by using aNanoDrop™ ND-1000 UV spectrophotometer (NanoDrop Technologies,Wilmington, Del.). RNA quality was assessed using a 2100 Bioanalyzerinstrument (Agilent Technologies, Palo Alto, Calif.) with the RNA 6000LabChip® kit (Agilent Technologies). Using the Agilent 2100 expertsoftware (Agilent Technology), RNA integrity number (RIN, AgilentTechnologies) was measured. In addition, RNA integrity was assessed byquality metric (QM) number, which is a novel metric for formalin-fixed,paraffin-embedded (FFPE) RNA. This metric is based on a weighedarea-under-the-curve approach.

Multiplex RT-PCR: Multiplex RT-PCR was performed using the MPCR kit(Maxim Biotech, San Francisco, Calif.) for mouse tumor necrosis factor(TNF) signaling genes set-3. This kit was designed to detect 9 genes,with amplicons ranging in size from 189 to 658 bp. Multiplex RT-PCR wascarried out according to the manufacturer's instructions in a total of50 μl reaction mixture. An initial pre-PCR step of 96° C. for 5 minuteswas performed in the Bio-Rad Icycler® PCR Thermal Cycler (Bio-Rad Lab,Hercules, Calif.), followed by a total of 37 PCR cycles under followingcondition: 2 cycles of 94° C. for 1 minute and 62° C. for 4 minutes andthen 35 cycles of 94° C. for 1 minute and 62° C. for 2 minutes. Thefinal cycle was followed by an additional incubation at 70° C. for 10minutes to complete partial polymerization. A MPCR positive control(Maxim Biotech) was used in each run. The positive control includedmouse frozen kidney RNA. A negative control containing no nucleic acidwas also included in each run to check for any PCR cross contamination.

An aliquot (1 μl) of multiplex RT-PCR product was loaded on the DNA 1000kit (Agilent Technologies, Palo Alto, Calif.) and capillaryelectrophoresed in the Agilent 2100 Bioanalyzer (Agilent Technologies).Agilent 2100 expert software (Agilent Technologies) was used forcomparison of electropherograms.

Real-time Quantitative RT-PCR: After removing the genomic DNA with DNAEliminator columns (Qiagen), 4 μg of total RNA were reverse transcribedinto first-strand cDNA using a QuantiTect® Reverse Transcription kit(Qiagen). cDNA samples were generated from each of three replicatesderived from different fixative solutions and frozen mouse kidney RNAand were used for quantitative real-time PCR using TaqMan® GeneExpression reagent (Applied Biosystems). Briefly, quantitative real-timePCR was performed with 2 μg of cDNA assayed in a 20 μl reaction volume.In order to assess RNA integrity, cycle threshold (Ct) value wasdetermined for 18S rRNA and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene. The experiment was carried out in triplicate. AMann-Whitney U test was used to evaluate the RNA integrity for eachfixative condition.

Assessment of DNA Quantity and Quality: DNA was extracted from a 1-mmtissue core using the QIAamp® DNA FFPE Tissue kit (Qiagen). Theextraction was performed according to the manufacturer's instructions.Prior to the final elution step, 40 μl of elution buffer was applied tothe column and incubated at room temperature for 2 minutes, followed bycentrifugation. The yields of DNA were determined using a NanoDrop™ND-1000 UV spectrophotometer (NanoDrop Technologies). The DNA qualitywas assessed using a BioScore™ Screening and Amplification kit (EnzoLife Sciences, Farmingdale, N.Y.) (Chung et al., Anal. Biochem.425:128-134, 2012). In addition, real-time PCR using TaqMan® GeneExpression reagent (Applied Biosystems) was performed. Briefly,quantitative real time PCR was performed with 1 μg of DNA assayed in a20 μl reaction volume. The reactions were incubated for 2 minutes at 50°C., for 10 minutes at 95° C. for initial denaturing and followed by 50cycles of 95° C. for 15 seconds and 60° C. for 1 minute in ABI 7500real-time PCR system (Applied Biosystems). In order to assess DNAintegrity, Ct value for HPRT gene was determined. The experiment wascarried out in triplicate. A Mann-Whitney U test was used to evaluatethe DNA integrity for each fixative condition.

Example 2 Histomorphology

This example describes histomorpholgic features of samples processedwith fixatives containing 70% ethanol, compared to NBF.

Fixatives are applied to mediate preservation of tissue, however intheir application for histopathology, the goal is retention of cyto- andhisto-morphologic features. FIG. 1 demonstrates the histomorphologicfeatures of tissue, with neutral buffered formalin (NBF) as thereference fixative, and the derivative fixatives of differentcombinations of 70% ethanol, PBS-buffered ethanol, and formulationscontaining glacial acetic acid and glycerol. 70% ethanol resulted ingreater shrinkage with poorer cytologic features, compared to NBF. Theintroduction of PBS as a buffering solution offset these changes, withless shrinkages and greater cytologic detail.

Phosphate-buffered ethanol fixatives were also tested in comparison toPBS buffered ethanol. Without being bound by theory, the presence ofsaline in the fixative appeared to contribute to cytomorphology quality,possibly by exerting osmotic effects. These differences appeared toresult in less cell and overall tissue shrinkage.

Example 3 Immunohistochemical Staining

This example describes evaluation of immunohistochemical staining ofsamples processed with fixatives containing 70% ethanol, compared toNBF.

Immunohistochemistry is very susceptible to differences in pre-analyticvariables, including fixation time and fixative type. The application ofantigen retrieval was originally performed to improve detection ofantigens in formalin fixed tissues by linearization of peptides, andchemical hydrolysis of protein-protein crosslinks, however it is nowapplied across preservation methods. Generally, tissues fixed withnon-crosslinking fixatives require less antigen retrieval.

FIG. 2 demonstrates immunohistochemistry for AQP1 and CD31 in mousekidney tissue sections. The staining pattern observed was optimized forEGAP fixation, and demonstrated the staining results under theseconditions for the other fixatives in the ethanol series, as well asNBF.

Example 4 Protein Quantity and Quality

This example describes evaluation of protein quality and quantityobtained from samples processed with fixatives containing 70% ethanol,compared to NBF.

Proteins were extracted from mouse kidney tissues fixed with thedifferent ethanol fixative formulations or NBF fixative, and subjectedto Western blotting employing anti-GAPDH antibodies. NBF fixed mousekidney tissue was used as a negative control. Although the proteinextraction yield of EGAP fixative showed the highest amount (5.25±0.490μg/mm³) among tested fixatives (FIG. 3A), there was no statisticallymeaningful difference between the different fixatives tested(4.42-5.25±0.918 μg/mm³).

The quality of protein extracted from the ethanol fixative formulationsand NBF fixed tissues was determined by Western blotting. Thequantitative image analyses of Western blot intensities were evaluatedby a Mann-Whitney U test. As shown in FIG. 3B, the GAPDH signal was morestrongly detected in the various ethanol fixed tissue samples (allp<0.001) than in NBF fixed tissue samples. In particular, GAPDH wasbetter preserved in EGAP-fixed tissue (approximately 32.6-fold increase)than in NBF fixed tissue, whereas EGP fixative (approximately 23.7-foldincrease) showed the lowest signals among tested ethanol fixatives.However, there was no statistically meaningful difference between theEGAP and the EGP fixative. These results suggest that the ethanolfixative formulations provide significant advantages in protein qualityover NBF, but did not significantly impact the amount of proteinrecovered.

Example 5 RNA Quantity and Quality

This example describes evaluation of RNA quality and quantity obtainedfrom samples processed with fixatives containing 70% ethanol, comparedto NBF.

RNA was extracted from mouse kidneys that were fixed with the differentfixatives under investigation and impregnated with paraffin. Thequantity of RNA was assessed by UV spectrophotometry. Compared to NBF(mean 3.32±0.15 EGAP (mean 7.35±0.69 μg/mm³) showed a significant effecton RNA recovery (P=0.005) (FIG. 4A). The 260/280 ratio of EGAP (mean2.01) was similar to NBF (mean 1.89).

Next, the quality of RNA extracted was analyzed by the 2100 Bioanalyzerinstrument (Agilent Technologies, Palo Alto, Calif.). Although therecovery of RNA from the EGAP fixative showed the highest quantity, theelectropherogram demonstrated that the RNA had limited lengths (FIG.4B). The pattern observed from FFPE tissue demonstrated shorterfragments between 100 and 200 nucleotides in length. In comparison, EGAPand the other ethanol fixatives showed increased RNA fragment length,supporting the finding that EGAP fixative preserved higher quality ofRNA than NBF.

The Ribosomal Integrity Number (RIN), which is the 28S to 18S rRNA,ratio has been widely adopted as a measure of RNA quality for RNAisolated from fresh and frozen tissue. The RIN remains an imperfectmeasure of quality, lacks strong correlation with gene-specificmeasurements (Schroeder et al., BMC Mol. Biol. 7:3, 2006), and cannot beaccurately applied to RNA isolated from formalin fixed, paraffinembedded tissue (Chung et al., J. Histochem. Cytochem 0.56:1033-1042,2008). In this context, we developed a quality metric (PERM—Paraffinembedded RNA Metric) number which is a novel metric for FFPE RNA. Thismetric is based on a weighed area-under-the-curve approach. Briefly,starting from an electropherogram (for example created by an Agilent2100 Bioanalyzer) or other methods of quantifying RNA fragment length(such as densitometry of an image of gel electrophoresis), a simplecalculation based on fluorescent units at specific time points was usedto qualify RNA integrity. The PERM provides a metric that placesprogressively greater value on the length of RNA in the calculatedmetric. The metric is calculated as follows:

QM=FU₂₅+(2*FU₃₀)+(3*FU₃₅)+(4*FU₄₀)+(5*FU₄₅)+(6*FU₅₀)+(7*FU₅₅)+(8*FU₆₀)+(9*FU₆₅)

where FU_(n) is fluorescence units at the indicated number of seconds.

As shown in FIG. 4C, the impact of individual components of theethanol-based fixatives on RNA quality can be dissected. The EGAP (BE70)fixative (mean PERM 123.40) and EAP fixative (mean PERM 121.23) showedthe highest numbers. The effect of PBS and glycerol addition on thisparticular metric was minimal. NBF showed the lowest number (mean PERM32.02) among those of the tested fixative conditions (FIG. 4C).

To further evaluate the quality of RNA extracted, RT-PCR with a smallsize amplicon (usually from 100 to 150 bp) was tested. To evaluate thesize limitation of amplicon of RNA extracted from tissue fixed with thepanel of fixatives and paraffin impregnated, multiplex RT-PCR using theMPCR kit for mouse TNF signaling genes set-3 was used. Four bands (205,235, 316, and 449 bp) that corresponded with the exact sizes of thetargets in ethanol fixatives were identified in the ethanol-fixedtissues, whereas two bands (235 and 316 bp) were detected in NBF fixedtissues with relatively weak signal (FIGS. 5A and 5B).

The effect of fixatives on RNA integrity was also evaluated by real-timequantitative RT-PCR using 18S rRNA and HPRT primers. Endogenous control18S rRNA generally showed low Ct-values (FIG. 5C), whereas HPRT geneshowed high Ct-values (FIG. 5D). The Ct-value of the qRT-PCRamplifications were 11.40 for 18S rRNA and 27.06 for HPRT inpreparations from fresh frozen (FF) tissues. Among the tested fixatives,EGAP resulted in Ct-values of 13.76 for 18S rRNA and 34.46 for HPRT,whereas EGP was demonstrated Ct values of 17.33 for 18S rRNA and 38.69for HPRT. In contrast, in samples generated from FFPE tissue theCt-values were higher, mean 25.06 and 41.27 for 18S rRNA and HPRT,respectively. It is clearly visible that the Ct-value of the EGAP wasthe lowest among all fixed samples, with 70% ethanol alone, EP and EAPshowing similar (though slightly higher) values (FIGS. 5C and 5D).

Example 5 DNA Quantity and Quality

This example describes evaluation of DNA quality and quantity obtainedfrom samples processed with fixatives containing 70% ethanol, comparedto NBF.

DNA was successfully extracted from all fixed tissues. The DNAextraction yield of EGAP was similar to that of NBF (mean 1.09-fold)(FIG. 6A). The 260/280 ratio of EGAP (mean 1.86) was also similar to NBF(mean 1.80). In addition, DNA obtained from evaluated fixed tissues wastested for array analysis suitability using BioScore™ Screening andAmplification Kit (Enzo Life Sciences, Farmingdale, N.Y.; Chung et al.,Anal. Biochem. 425:128-134, 2012). Using a 100 ng DNA template extractedfrom each of the ethanol-fixed tissues, approximately 9.73±0.23 μg ofDNA of good quality was amplified for nucleic acid array analysis.However, the DNA prepared from NBF fixed tissue showed intermediatequality (mean 2.81±0.13 μg) for the microarray application (FIG. 6B).

To evaluate the impact of different fixatives on DNA integrity,real-time PCR using HPRT primers was carried out. As shown in FIG. 6C,Ct-value of EGAP fixed tissue (mean Ct 14.70) was lower than that of NBF(mean Ct 21.25). In addition, the Ct value of EGAP fixed tissue wassimilar to 70% ethanol as a fixative (mean Ct 15.11). As expected, theCt value of fresh frozen (mean Ct 11.25) tissue was lower than that ofethanol-fixed or NBF-fixed tissue (FIG. 6C).

Example 6 Varying Fixative Component Concentrations

This Example describes fixatives with 70% ethanol and varying amounts ofPBS, glycerol, and acetic acid.

Fixative formulations including 70% ethanol, 0.2-1×PBS, 1-5% glycerol,and 0-10% glacial acetic acid. Tables 2-4 show the compositionsproduced. Compositions that did not precipitate were tested by fixingtissue and evaluating tissue staining (H&E), quality of RNA (by PCRand/or QM), and immunohistochemistry.

TABLE 2 Fixatives with 70% ethanol, 0.5X PBS and varying glycerol andacetic acid Glacial Acetic 10N EtOH PBS Glycerol Acid starting Room NaOHFinal Room (%) (X) (%) (%) pH Comments 4° C. O/N Temp (μL) pH Comments4° C. O/N Temp 70 0.5 1 0 8.1 soln. cloudy precipitant precipitant <8but >7 pH no @ RT change before after 4° acetic acid but was addedprecipitant still remains 70 0.5 2 0 8.12 soln. cloudy precipitantprecipitant <8 but >7 pH no @ RT change before after 4° acetic acid butwas added precipitant still remains 70 0.5 5 0 8.15 soln. cloudyprecipitant precipitant <8 but >7 pH no @ RT change before after 4°acetic acid but was added precipitant still remains 70 0.5 1 1 4.45soln. cloudy no no ~350 7.02 soln precipitant @ RT precipitantprecipitant remain before clear acetic acid was added 70 0.5 1 2.5 4.09soln. cloudy no no >900 7 soln precipitant @ RT precipitant precipitantremain before clear acetic acid was added 70 0.5 1 5 3.81 soln. cloudyno no >1000 vol. of @ RT precipitant precipitant NaOH before >1 ml, noacetic acid further was added testing needed 70 0.5 1 10 3.45 soln.cloudy no no >1000 vol. of @ RT precipitant precipitant NaOH before >1ml, no acetic acid further was added; testing glycerol needed dropletspresent 70 0.5 2 1 4.47 soln. cloudy no no <1000 7.03 precipitantprecipitant @ RT precipitant precipitant before acetic acid was added 700.5 2 2.5 4.08 soln. cloudy no no >1000 7.05 soln no @ RT precipitantprecipitant remain precipitant before clear acetic acid was added 70 0.52 5 3.78 soln. cloudy no no vol. of @ RT precipitant precipitant NaOHbefore >1 ml, no acetic acid further was added testing needed 70 0.5 210 3.44 soln. cloudy no no vol. of @ RT precipitant precipitant NaOHbefore >1 ml, no acetic acid further was added; testing glycerol neededdroplets present 70 0.5 5 1 4.41 soln. cloudy no no 7.1 soln little @ RTprecipitant precipitant remain precipitant before clear acetic acid wasadded 70 0.5 5 2.5 4.23 soln. cloudy no no >1000 7.01 soln no @ RTprecipitant precipitant remain precipitant before clear acetic acid wasadded 70 0.5 5 5 3.75 soln. cloudy no no vol. of @ RT precipitantprecipitant NaOH before >1 ml, no acetic acid further was added; testingglycerol needed droplets present 70 0.5 5 10 3.41 soln. cloudy no novol. of @ RT precipitant precipitant NaOH before >1 ml, no acetic acidfurther was added; testing glycerol needed droplets present

TABLE 3 Fixatives with 70% ethanol and varying PBS, glycerol, and aceticacid (total volume 100 ml) Glacial Glacial Acetic Heat @ Acetic 10N EtOHstarting Acid 65° C. (~1- Acid NaOH (%) PBS Glycerol pH (μL) pH 4° C.O/N 1.5 hr) (μL) pH pH 4° C. O/N 70  1X 1% 7.67 120 4.84 no no 0 4.885.10 no precipitant precipitant 70  1X 2% 7.68 120 4.88 no no 0 4.855.07 no precipitant precipitant 70  1X 5% 7.71 120 4.89 no no 0 4.875.03 no precipitant precipitant 70 0.5X 1% 8.10 100 4.8 no no 0 4.775.10 no precipitant precipitant 70 0.5X 2% 8.10 100 4.77 no no 0 4.755.02 no precipitant precipitant 70 0.5X 5% 8.11 100 4.72 no no 0 4.785.00 no precipitant precipitant 70 0.29X  1% 8.48 0 precipitant Glycerol40 4.95 5.07 no present precipitant 70 0.28X  2% 8.47 0 precipitantGlycerol 40 4.93 5.05 no present precipitant 70 0.25X  5% 8.45 0precipitant Glycerol 30 5.05 0.00 no present precipitant

TABLE 4 Fixatives with 70% ethanol, 0.5X PBS, 5% glycerol, and varyingacetic acid (total volume 100 ml) Glacial Acetic 10N starting Acid NaOHEtOH PBS Glycerol pH (μL) pH (μL) pH 4° C. O/N Comments 70% 0.5X 5% 8.21100 5.12 0 5.12 no no precipitant precipitant @ RT 70% 0.5X 5% 8.25 5004.44 ~80 5.00 no no precipitant precipitant @ RT 70% 0.5X 5% 8.22 10004.13 ~120 5.01 no no precipitant precipitant @ RT 70% 0.5X 5% 8.20 15003.95 ~230 5.01 no no precipitant precipitant @ RT

Example 7 Effect of Fixation Time with BE70

This example compares the effect of length of fixation of tissue in BE70and NBF on histomorphology, IHC, Western blotting, and protein recovery.

Mouse, liver and kidney tissue was fixed in BE70 or NBF for varyingamounts of time from 4 hours to 6 months to determine performance ofhistology, IHC, Western blotting, and protein recovery at various timepoints.

IHC of mouse kidney tissue with Aquaporin (FIG. 7A) or Ki-67 (FIG. 7B)showed that BE70 fixation provides adequate quality at 4 hours offixation and this is retained to at least 3 months fixation. Incontrast, fixation with NBF only provides adequate quality from about 1day to 1 week.

Protein recovery (FIG. 8A) and Western blotting (FIGS. 8B and 8C) wasgenerally best with BE70 fixation for about 12 hours to one month. NBFfixation was generally best for Western blotting at 12 hours to 1 day.

Finally, histomorphology (H&E staining; FIG. 9) was evaluated for BE70and NBF at various times. BE70 provided good results from at least 12hours to 6 months fixation, and was only slightly less effective thanNBF at 4 hours. In contrast NBF fixation was inadequate at 4 hours,adequate for 12 hours to 1 day, and dropped out by 1 week.

Example 8 BE70 Fixative with Guanidinium Salts

This example describes the effect of adding guanidinium salts to theBE70 fixative.

BE70 solutions (70% ethanol (v/v), 0.5×PBS (v/v), 1% glycerol (v/v), and0.5% glacial acetic acid (v/v)) were prepared as described above, withthe addition of guanidinium salts. Guanidine HCl was added at a finalconcentration of 0.8 M or 1.6 M. Guanidine thiocyanate (GT) was added at0.51 M, 1.02 M, or 1.69 M. Guanidine acetate salt (GAS) was added at0.84M GAS (I) and 0.42M GAS (II). Mouse liver and kidney tissue sampleswere collected. In order to examine the impact of the fixatives incomparison to 10% NBF in antigen degradation and molecular quality,mouse kidney samples were fixed for 24 hours at room temperature in 10ml of the different fixatives. Tissues were then processed using anenclosed automated processor (Tissue-Tek VIP IV, Sakura Finetek Inc.,Torrance, USA) at roughly 30-45 minutes per station. Briefly, tissueswere dehydrated in a series of ethanol, then cleared with xylene priorto infiltration with molten paraffin. Tissues were then embedded inparaffin and sectioned for histological and molecular evaluation.Molecular evaluation was performed as described in Example 1.

Western blotting of kidney tissue fixed with BE70 plus guanidine HCl orGT showed improved detection of AKT compared to BE70 alone or NBF (FIG.10). BE70+GT appeared to be superior to BE70+guanidine HCl. This issupported by additional data below.

The paraffin embedded RNA metric (PERM, previously referred to asQuality Metric) was evaluation for BE70+GT. The PERM is a measure or RNAquality applied to paraffin embedded tissue, that has high (0.9)correlation with gene specific metrics (see, e.g., Chung et al.,Biotechniques. 60(5):239-44, 2016). The BE70+GT solutions demonstratedsuperior performance (FIG. 11).

The effect of the different fixatives on RNA integrity was alsoevaluated by real-time quantitative RT-PCR using TLR-4 (FIGS. 12A-12C)and ACTB (FIG. 13A-13C) primers. The BE70+GT showed improved performancecompared to NBF and equivalent or improved performance compared to BE70, at least at the two higher concentrations of GT.

Western blotting and protein recovery were evaluated for liver andkidney samples fixed in BE70-GT (1.69 M GT) or BE70+GAS (FIGS. 14A and14B). As demonstrated in FIGS. 14A and 14B, the presence of guanidiniumin the fixative improved both the quantity of protein recovered (FIG.14B) but when loaded into a western blot on an equal proteinconcentration, the detection of full length proteins by western blot wasenhanced win the presence of guanidinium compared to both BE70 and NBF.This increase in both total protein quantity recovered, and theimmunodetection of proteins (both AKT and GAPDH) when normalized fortotal protein loaded on the western blot is seen with guanidiniumthiocyanate and guanidinium acetate, and in both instances occurs in aconcentration depended manner, with higher concentrations of guanidiniumassociated with more protein recovered, and improved immunodetection.

The addition of guanidinium displaces unfreezable/non-freezable water,resulting a decrease in the degradation of full length peptides andnucleic acids. This displacement of unfreezable/non-freezable waterresults in the improved stability of the biospecimen, preventinghydrolysis and oxidation in the paraffin embedded block.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

We claim:
 1. A fixative comprising ethanol and at least one guanidiniumsalt.
 2. The fixative of claim 1, wherein the at least one guanidiniumsalt is selected from guanidinium HCl, guanidinium thiocyanate,guanidinium acetate, guanidinium sulfate, guanidinium nitrate,guanidinium nitrate, guanidinium carbonate, guanidinium formate andguanidinium phosphate.
 3. The fixative of claim 2, wherein the at leastone guanidinium salt is guanidinium thiocyanate.
 4. The fixative ofclaim 1, wherein the fixative further comprises a phosphate buffer. 5.The fixative of claim 4, wherein the phosphate buffer isphosphate-buffered saline.
 6. The fixative of claim 5, wherein thefixative comprises 70% ethanol, 0.5× phosphate-buffered saline and atleast one guanidinium salt.
 7. The fixative of claim 1, wherein thefixative does not contain added aldehyde-containing compounds.
 8. Thefixative of claim 1, wherein the fixative further comprises at least onealdehyde-containing compound.
 9. The fixative of claim 8, wherein the atleast one aldehyde-containing compound is formaldehyde,paraformaldehyde, or glutaraldehyde.
 10. The fixative of claim 1,further comprising at least one of: ethylenediaminetetraacetic acid orethylene glycol-bis(β-aminoethyl ether) ethylenediaminetetraacetic acid;trichloroacetic acid; trehalose; acrylamide; polyethylene glycol;ethylene glycol; and acetonitrile.
 11. A method, comprising immersing acell or tissue sample in the fixative of claim 1 under conditionssufficient to fix the cell or tissue sample.
 12. The method of claim 11,wherein conditions sufficient to fix the cell or tissue compriseimmersing the sample in the fixative at room temperature for at least 12hours.
 13. The method of claim 11, further comprising processing thefixed sample, wherein the processing comprises: dehydrating the fixedsample; and embedding the dehydrated fixed sample in an embeddingmedium.
 14. The method of claim 13, wherein the embedding mediumcomprises paraffin or a resin.
 15. The method of claim 11, furthercomprising analyzing histomorphology of the fixed sample and/orpresence, amount, or quality of RNA, DNA, and/or protein in the fixedsample.
 16. A method, comprising immersing a cell or tissue sample inthe fixative of claim 8 under conditions sufficient to fix the cell ortissue sample.
 17. The method of claim 16, wherein conditions sufficientto fix the cell or tissue comprise immersing the sample in the fixativeat room temperature for at least 12 hours.
 18. The method of claim 16,further comprising processing the fixed sample, wherein the processingcomprises: dehydrating the fixed sample; and embedding the dehydratedfixed sample in an embedding medium.
 19. The method of claim 18, whereinthe embedding medium comprises paraffin or a resin.
 20. The method ofclaim 16, further comprising analyzing histomorphology of the fixedsample and/or presence, amount, or quality of RNA, DNA, and/or proteinin the fixed sample.